The Cloning Of Myosin 7a Gene And Functional Analysis In Gampsocleis Gratiosa

Myosin is a class of micromilament-dependent motor protein superfamily,which consists of the head motor,neck and tail of the N-terminal.Myosin ?A,a member of myosin ? family,is expressed in many organs and is a "cargo" transporter.Myosin ?a is also a common component of cilia and microvilli.Gampsocleis gratiosa is widely distributed in the north of China and easy to obtain.It is a good material to study the molecular and cellular biological mechanism of Orthoptera.The functional analysis of Myosin ?A can not only supplement the information of insect cell development,but also lay a molecular foundation for the study of reproduction,digestion and excretion of Orthoptera insects and the control of Orthoptera pests.Firstly,the transcriptome database of Gampsocleis gratiosa adults was sorted out,and the myosin family members in the database were analyzed.Based on the transcriptome database of Gampsocleis gratiosa adults,the myosin family was retrieved and the myosin was identified by NCBI Blastp online comparison.Seven myosin genes were identified in the transcriptome: GgMyo1 b,GgMyo5 a,GgMyo6,GgMyo7a,GgMyo9 a,GgMyo18 a and GgMyo19,respectively.The tail domain is complex,consisting of two repeated My TH4-FERM composite structures in series,between SH3 structures;The GgMyo7a gene was cloned and identified by bioinformatics.The full-length sequence of GgMyo7a gene is8938 bp,which contains an open reading frame encoding 1865 amino acids with a molecular weight of 216.18 KD.The domain of GgMyosin ?A: a head motor is followed by the neck of four IQ motifs;the tail domain is complex,which is composed of two repeated My TH4-FERM domain in series,with SH3 structure in the middle.GgMyosin ?A structure is also consistent with the structure Myosin ?A known species.Then,the RNA interference technology was used to knock down the Myo7a at mRNA level.The results of q PCR in different organs showed that the expression of Myo7a was higher in testis,digestive organs(gastric caecum,midgut)and excretory organs(malpighian canal).The interference fragment corresponding to the target fragment of GgMyo7a was designed,and the synthesized Myo7a double stranded interference RNA(dsmyo7a)was injected in vitro by RNA interference technology.The results of q PCR quantitative analysis showed that the optimal dosage of dsmyo7 a was 50?g,and the expression of Myo7a was down regulated at mRNA level after injection of dsmyo7 a,and the expression of Myo7a was the lowest at the fifth day,about 80% down.Finally,dsmyo7 a was injected in vitro.At the cell level,the effects of RNA interference and knockdown of Myo7a on Spermiogenesis and microvilli of digestive tract and malpighian tube epithelial cells were observed and analyzed by H&E staining and PAS-hematoxylin of paraffin section,immunofluorescence staining of frozen section,U-Pb double staining of ultra-thin section.The results showed that during the eight stages of Spermiogenesis,no obvious changes were observed in the nucleus,acrosome,microfilament and microtubule of the testis after the interference.After the interference,the microvilli of gastric caecum,midgut and malpighian tube epithelial cells were loosely arranged,and the edge was discontinuous;the morphology of microvilli monomer changed,and the monomer membrane was also obviously loose.In addition,the peritrophic membrane of midgut was destroyed.Comprehensive analysis of RNA interference results: after RNA interference,the distribution of microvilli in gastric caecum,midgut and malpighian tube epithelial cells was loose,the edge boundary was discontinuous,and the morphology of microvilli monomer changed.These results indicate that myosin ?a plays an important role in the maintenance of microvilli finger like processes in the digestive tract and Malpighian tube epithelial cells.The cross section of microvilli in the midgut epithelial cells after interference was observed by transmission electron microscope,and it was found that the membrane of microvilli monomer was obviously loose,which further speculates that MYO7A may play a role between the microfilament and membrane of microvilli monomer.